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- EMDB-5783: Bacteriophage-encoded Tubulin Homologue PhuZ Forms a Three-Strand... -

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Basic information

Entry
Database: EMDB / ID: EMD-5783
TitleBacteriophage-encoded Tubulin Homologue PhuZ Forms a Three-Stranded Filament
Map dataReconstruction of PhuZ filament structure
Sample
  • Sample: PhuZ201 filament
  • Protein or peptide: PhuZ
KeywordsPhuZ / FtsZ/tubulin-related protein / filament / helical reconstruction / Pseudomonas bacteriophages / 201phi2-1
Function / homologyestablishment of localization / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / host cell cytoplasm / GTPase activity / GTP binding / identical protein binding / Phage tubulin-like protein
Function and homology information
Biological speciesPseudomonas phage 201phi2-1 (virus)
Methodhelical reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsZehr EA / Kraemer JA / Erb ML / Coker JKC / Montabana EA / Pogliano J / Agard DA
CitationJournal: Structure / Year: 2014
Title: The structure and assembly mechanism of a novel three-stranded tubulin filament that centers phage DNA.
Authors: Elena A Zehr / James A Kraemer / Marcella L Erb / Joanna K C Coker / Elizabeth A Montabana / Joe Pogliano / David A Agard /
Abstract: Tubulins are a universally conserved protein superfamily that carry out diverse biological roles by assembling filaments with very different architectures. The underlying basis of this structural ...Tubulins are a universally conserved protein superfamily that carry out diverse biological roles by assembling filaments with very different architectures. The underlying basis of this structural diversity is poorly understood. Here, we determine a 7.1 Å cryo-electron microscopy reconstruction of the bacteriophage-encoded PhuZ filament and provide molecular-level insight into its cooperative assembly mechanism. The PhuZ family of tubulins is required to actively center the phage within infected host cells, facilitating efficient phage replication. Our reconstruction and derived model reveal the first example of a three-stranded tubulin filament. We show that the elongated C-terminal tail simultaneously stabilizes both longitudinal and lateral interactions, which in turn define filament architecture. Identified interaction surfaces are conserved within the PhuZ family, and their mutagenesis compromises polymerization in vitro and in vivo. Combining kinetic modeling of PhuZ filament assembly and structural data, we suggest a common filament structure and assembly mechanism for the PhuZ family of tubulins.
History
DepositionOct 31, 2013-
Header (metadata) releaseDec 25, 2013-
Map releaseMar 26, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.385
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.385
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j5v
  • Surface level: 0.385
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j5v
  • Surface level: 0.385
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j5v
  • Imaged by Jmol
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5783.gif

Downloads & links

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Map

FileDownload / File: emd_5783.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of PhuZ filament structure
Voxel sizeX=Y=Z: 1.20398 Å
Density
Contour LevelBy AUTHOR: 0.385 / Movie #1: 0.385
Minimum - Maximum-2.62722111 - 3.01486778
Average (Standard dev.)-0.00029646 (±0.17518339)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 240.79599 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.203981.203981.20398
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z240.796240.796240.796
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-2.6273.015-0.000

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Supplemental data

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Sample components

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Entire : PhuZ201 filament

EntireName: PhuZ201 filament
Components
  • Sample: PhuZ201 filament
  • Protein or peptide: PhuZ

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Supramolecule #1000: PhuZ201 filament

SupramoleculeName: PhuZ201 filament / type: sample / ID: 1000 / Oligomeric state: filament / Number unique components: 1

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Macromolecule #1: PhuZ

MacromoleculeName: PhuZ / type: protein_or_peptide / ID: 1 / Name.synonym: PhuZ201 / Oligomeric state: polymer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas phage 201phi2-1 (virus) / synonym: bacteriophage
Molecular weightExperimental: 34.621 KDa / Theoretical: 34.621 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28a(+)
SequenceUniProtKB: Phage tubulin-like protein / InterPro: Tubulin/FtsZ, GTPase domain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 8
Details: 50 mM HEPES, 125 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM GMPCPP
GridDetails: C-FLAT holey carbon grid, 200 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK III
Method: Sample was applied to C-FLAT holey carbon grids, blotted for 2 seconds, and flash frozen in liquid ethane

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at 250,000 times magnification
DateNov 21, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 461 / Average electron dose: 25 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Whole micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 14.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 116.4 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.1 Å / Resolution method: OTHER / Software - Name: Spider
DetailsReconstruction was determined by IHRSR.

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Atomic model buiding 1

Initial modelPDB ID:

3r4v


Chain - Chain ID: A
SoftwareName: Chimera, Coot
DetailsFlexible fitting applied to the C-terminus (residues 272-315)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross correlation
Output model

PDB-3j5v:
PhuZ201 filament

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